牛睾丸支持细胞分离、纯化及体外生长行为的研究

了解牛睾丸支持细胞体外培养的生物学特性,试验采用组合酶消化和选择贴壁法,将5月龄、6月龄牛胎儿及新生牛睾丸支持细胞分离纯化后进行体外培养。试验结果显示,这一阶段牛睾丸适宜支持细胞分离纯化用;采用0.25 %胰蛋白酶+0.02 %EDTA二次消化法是一种经济有效的牛胎儿睾丸支持细胞分离方案;试验发现,牛胎儿睾丸支持细胞体外研究时间应控制在3天～20天内,处于对数生长期的牛胎儿睾丸支持细胞,对牛体外受精卵体外发育有明显的促进作用。     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Binding of 125I-insulin-like growth factor-II to cells cultured in fetal bovine serum: a complication

Insulin-like growth factor II is an important fetal mitogen in mice and humans and its biological activity is regulated in a complex manner. The peptide interacts with three membrane-bound receptors, with a superfamily of insulin-like growth factor binding proteins and with the proteoglycan, glypican-3. Recently, the blood protein, vitronectin, has been identified as a novel insulin-like growth factor II-binding protein. Many studies have used cell lines maintained in fetal bovine serum to identify cell surface insulin-like growth factor II binding sites. We now describe a complication associated with the interpretation of such in vitro studies. Fetal bovine serum-derived vitronectin adheres very tightly to tissue culture dishes. When cells that have been maintained in fetal bovine serum are incubated with 125I-insulin-like growth factor II, a substantial fraction of the 125I-insulin-like growth factor II apparently associated with the cell surfaces may represent radioliogand bound by the fetal bovine serum-derived vitronectin. This may result in over-estimation of cell surface insulin-like growth factor II binding sites.     

Bovine fetal fluid cells in vitro: fate and fetal sex prediction accuracy

The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex predication from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70 percent of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis whereas only 10 percent of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100 percent in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.     

Bovine fetal fluid cells in vitro: Fate and fetal sex prediction accuracy

Summary The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex prediction from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70% of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis, whereas only 10% of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100% in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.     

Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term and association with high birth weights

This study was designed to characterize conceptus development based on pre- and postnatal measurements of in vivo- and in vitro-derived bovine pregnancies. In vivo-produced embryos were obtained after superovulation, whereas in vitro-produced embryos were derived from established procedures for bovine IVM, IVF and IVC. Blastocysts were transferred to recipients to obtain pregnancies of single (in vivo/singleton or in vitro/singleton groups) or twin fetuses (in vitro/twins group). Ultrasonographic examinations were performed weekly, from Day 30 of gestation through term. Videotaped images were digitized, and still-frames were used for the measurement of conceptus traits. Calves and fetal membranes (FM) were examined and measured upon delivery. In vitro-produced fetuses were smaller than in vivo controls (P < 0.05) during early pregnancy (Day 37 to Day 58), but in vitro/singletons presented significantly higher weights at birth than in vivo/control and in vitro/twin calves (P < 0.05). From late first trimester of pregnancy (Day 72 to Day 93), placentomes surrounding in vitro-derived singleton fetuses were longer and thinner than controls (P < 0.05). At term, the presence of giant cotyledons in the fetal membranes in the in vitro group was associated with a larger cotyledonary surface area in the fetal horn (P < 0.05). The biphasic growth pattern seen in in vitro-produced pregnancies was characterized by conceptus growth retardation during early pregnancy, followed by changes in the development of the placental tissue. Resulting high birth weights may be a consequence of aberrant placental development due to the disruption of the placental restraint on fetal growth toward the end of pregnancy.     

Long-term culture of human endothelial cells

Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10(3)/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.     

Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Human and bovine vascular endothelial cells. Comparative effect on cell growth and longevity of an eye-derived growth factor (EDGF) and of extracellular matrix

Human and bovine vascular endothelial cells from the umbilical vein and the aorta, respectively, were cultured in the presence of EDGF (a growth factor prepared from bovine retina) on plastic or on extracellular matrix (ECM). Both EDGF and ECM are required to allow the maximal proliferation of human cells and their organization in a typical monolayer. Conversely, bovine aortic endothelial cells grow perfectly in the absence of both factors in 6% fetal calf serum. However, a requirement for EDGF can also be demonstrated in low serum conditions, or in cells at high passage number. ECM had no growth promoting activity by itself. Thrombin acts similarly to EDGF on bovine serum-starved cells. EDGF prolongs the in vitro lifespan of both types of cells. Cells at all stages still synthesize factor VIII antigen as revealed by immunofluorescence. Thus EDGF, like other growth factors from brain, FGF or ECGF, may have an important role in angiogenesis, a critical problem in pathological retinas.     

Effect of fractions of horse and fetal bovine serum on neoplastic conversion of C3H mouse cells in tissue culture

Summary Cultures derived from C3H/He mouse embryos were grown in medium NCTC 135 supplemented with horse serum, fetal bovine serum, or various combinations of large and small molecule fractions of horse and fetal bovine serum. Cultures in medium NCTC 135 alone or in medium 135 supplemented with the small molecule fraction of either horse or fetal bovine serum did not grow as continuous long term lines. The best growth was obtained when the cultures were in medium containing the large molecule fraction of fetal bovine serum either alone or in combination with a small molecule fraction. Cells grown in the presence of the low molecular weight fraction of horse serum invariably produced tumors on injection into syngeneic animals. Cells in the small molecular weight fraction of fetal bovine serum combined with the large molecular weight fraction of horse serum produced tumors after a prolonged period in vitro. *** DIRECT SUPPORT *** A00S8010 00003     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Proliferative characteristics of clonal endothelial cell strains

We have utilized clonal strains of bovine fetal aortic endothelial cells to study cellular senescence in a differentiated cell type of physiological significance. Serial subcultivation of nine endothelial clones derived from three fetal calf aortas revealed proliferative life-spans in vitro of 53–125 population doublings (PDs), compared with 60 and 143 PDs for two lines of bovine fetal lung cells and 85 and 147 PDs for two lines of bovine vascular smooth muscle cells. Serial growth curves showed marked reductions associated with endothelial cellular senescence both in cellular growth rate and culture plateau density. Studies of the 24-hour [³H]-thymidine labeling index versus percentage of proliferative life-span completed indicated that clonal endothelial cultures contained a large proportion (greater than 90%) of rapidly cycling cells until about 75% of the life-spans were completed. Senescent endothelial cells showed evidence of large increases in cell area, cell volume, and protein content. In those clones examined, one specialized endothelial function, Factor VIII antigen expression, was retained qualitatively throughout the life-spans.     

Age-related and carcinogen-induced alterations of the extracellular growth factor requirements for cell proliferation in vitro

Cells from thigh muscles of fetal rats proliferate readily in vitro in medium containing homologous adult rat "plasma". As the donor animals mature, these cells become unable to make DNA and proliferate in "plasma" medium, but retain an ability to proliferate in medium containing fetal bovine serum (FBS). This age-related loss of the ability to proliferate in "plasma"-medium is due to an increasing need by the cells for an exogenous prereplicative promoter which is found in FBS and a crude preparation of bovine luteinizing hormone. Adult cells (and possibly fetal cells) also require a "cycle-completion" factor which is found in FBS and adult rat "plasma". The requirements for such external proliferative promoters is completely eliminated by neoplastic transformation in vivo, and neoplastic adult cells isolated from a nickel sulfide-induced mixed rhabdomyo-fibro-sarcoma can make DNA and proliferate in vitro in the complete absence of exogenous growth factors.     

Post hatching development: a novel system for extended in vitro culture of bovine embryos

Although acceptable rates of blastocyst formation are achieved with in vitro production of bovine embryos, several problems still compromise the subsequent development of the fetus and newborn, especially in embryos originating from somatic cell nuclear transfer. Routinely, the potential development of a bovine conceptus is predicted either on blastocyst quality or on various parameters related to the embryonic-fetal development in a foster mother. These methods are either imprecise or costly, highlighting the need for more reliable and practical methods to evaluate early embryonic development and differentiation. Thus, our aim was to improve the in vitro culture of embryos post hatching and to define a stable and repeatable system to monitor the development of bovine embryos. For that, in vitro-derived embryos were cultured in agarose gel tunnels in a modified culture medium (SOFaaci within 10% fetal bovine serum and 27.7 mM glucose). Daily monitoring of embryo length revealed that 56%-67% of the embryos in culture showed rapid growth and elongated until Day 13. Electron microscopy of elongated embryos at Day 14 confirmed successful localization of differentiated cells forming the trophoblast and hypoblast, with the definition of the Rauber layer. In conclusion, a stable culture system of post hatching embryos was first defined and can be used as a model for rapid growth, elongation, and initial differentiation of bovine post hatching embryos produced entirely in vitro.     

The effect of human follicular fluid on endothelial cells: proliferation and DNA synthesis

Human follicular fluid has been reported to cause angiogenesis. Although endothelial cell mitogenesis is a major component of the process of angiogenesis, the findings in the literature regarding the effects of human follicular fluid in in vitro endothelial cell growth assays are equivocal. In the present study, we examined the effect of human follicular fluid from preovulatory follicles on fetal bovine aortic endothelial cell proliferation. Human serum was used as a control since follicular fluid is largely a transudate of serum and could contain serum-derived endothelial cell mitogens. Neither human follicular fluid nor serum directly caused endothelial cell proliferation. However, follicular fluid, as well as serum, caused an increase in thymidine incorporation by endothelial cells, and resulted in an increased proportion of cells in the DNA synthesis and G2 phases of the cell cycle. Although follicular fluid was not directly mitogenic, it, in contrast to human serum, together with fetal bovine serum markedly enhanced endothelial cell proliferation beyond that caused by fetal bovine serum alone. These results suggest that a combination of factors, some of ovarian origin present in follicular fluid, and others from as yet unidentified sources, govern the mitogenic component of new blood vessel growth in the ovary.     

A cartilage growth mixture model with collagen remodeling: validation protocols

A cartilage growth mixture (CGM) model is proposed to address limitations of a model used in a previous study. New stress constitutive equations for the solid matrix are derived and collagen (COL) remodeling is incorporated into the CGM model by allowing the intrinsic COL material constants to evolve during growth. An analytical validation protocol based on experimental data from a recent in vitro growth study is developed. Available data included measurements of tissue volume, biochemical composition, and tensile modulus for bovine calf articular cartilage (AC) explants harvested at three depths and incubated for 13 days in 20% fetal borine serum (FBS) and 20% FBS+beta-aminopropionitrile. The proposed CGM model can match tissue biochemical content and volume exactly while predicting theoretical values of tensile moduli that do not significantly differ from experimental values. Also, theoretical values of a scalar COL remodeling factor are positively correlated with COL cross-link content, and mass growth functions are positively correlated with cell density. The results suggest that the CGM model may help us to guide in vitro growth protocols for AC tissue via the a priori prediction of geometric and biomechanical properties.